The submersed monocot, Hydrilla verticillata (L.f.) Royle, is a facultative C(4) NADP-malic enzyme (NADP-ME) plant in which the C(4) and Calvin cycles co-exist in the same cell. Futile cycling is avoided by an intracellular separation of carboxylases between the cytosol and chloroplasts. Of the two sequenced H. verticillata phosphoenolpyruvate carboxylase (PEPC) isoforms, hvpepc3 and hvpepc4, transcript expression of the latter was substantially up-regulated during C(4) induction, especially in the light. Western blots revealed two PEPC-specific bands in C(3) and C(4) leaf extracts; the lower band dominated in the C(4) and underwent post-translational phosphorylation in the light as determined by immunological studies. This band probably represents the photosynthetic isoform, HVPEPC4, despite the lack of the C(4) signature serine (Flaveria residue 774; Hydrilla 779). In C(4) leaves, PEPC activity increased 14-fold, was enhanced by leaf exposure to light, and showed allosteric regulation. Glucose-6-phosphate acted as a positive effector, but malate was inhibitory, with I(50) values of 0.4 and 0.2 mM in the light and dark, respectively, similar to those of other C(4) PEPC isoforms. In contrast, in C(3) leaves, transcript expression of both isoforms was weak, with little evidence of diel regulation, and the PEPC proteins showed essentially no indication of phosphorylation. PEPC activity in C(3) leaves was low, light independent and followed Michaelis-Menten kinetics. It was tolerant to malate, with 10-fold higher I(50) values than the PEPC from C(4) leaves. These data suggest that hvpepc4 encodes the C(4) photosynthetic PEPC, and hvpepc3 encodes an anaplerotic form.